Expression of Microbial Genes Involved in the Reductive Dehalogenation of PCE and its Degradation Products in a PCE-Contaminated, Constructed WetlandEPA Grant Number: F6D70518
Title: Expression of Microbial Genes Involved in the Reductive Dehalogenation of PCE and its Degradation Products in a PCE-Contaminated, Constructed Wetland
Investigators: Isenhouer, Gwyn
Institution: Wright State University - Main Campus
EPA Project Officer: Zambrana, Jose
Project Period: September 1, 2006 through September 1, 2008
Project Amount: $111,172
RFA: GRO Fellowships for Graduate Environmental Study (2006) RFA Text | Recipients Lists
Research Category: Biology/Life Sciences , Ecological Indicators/Assessment/Restoration , Academic Fellowships , Fellowship - Environmental Science , Fellowship - Microbiology , Hazardous Waste/Remediation
Tetrachloroethene (PCE), and its degradation products, trichloroethene (TCE), isomers of dichloroethene (DCE), and vinyl chloride (VC) are common groundwater contaminants that pose health risks to humans. PCE and its daughter products are most efficiently degraded by a microbial consortium utilizing a combination of oxidative, co-metabolic and reductive, dehalorespiring pathways. Microbes catalyze the latter through the expression of reductive dehalogenase (RDase) enzymes that are specific for one or two of the VOC substrates. The objective of this project is to determine whether the levels of RDase gene expression can be correlated with the disappearance of the respective VOC substrates within a PCE-contaminated, constructed wetland. Towards this end, we will develop real-time, reverse transcriptase polymerase chain reaction (RT-PCR) assays to monitor these genes, and test the utility of these assays for field application and monitoring.
Currently, the microbial community within the wetland is being characterized through the construction of a 16S rRNA clonal library. Nested PCR has been used to identify known reductively-dehalogenating microbes. These data contribute to our ability to predict RDases that may be operating at this site. To find these enzymes, we are optimizing PCR conditions with degenerate RDase primers for detection of the genes in a known reductively-dehalogenating culture as a control, as well as in DNA extracts from soils samples of the contaminated wetland. The genes identified in the wetland will be the targets for development of RT- PCR assays in three stages: development and optimization of the assays against pure DNA and positive control cultures, testing of the assays against lab-scale microcosms, and testing of the assays in field-study applications. RDase expression levels will then be compared to VOC substrate concentrations collected by collaborators working at the same site.
We hope to determine the lowest-threshold concentration of each contaminant for which expression of its specific RDase is first detected. Likewise, the maximum quantity of RDase expression, above which further degradation is not accelerated, will be a significant finding. These limits will allow us to predict the range of contaminant concentrations and gene expression that must exist for optimum degradation through reductive dehalogenation. Assays developed in this study will help drive cost-efficient engineering and operation of treatment systems that include bioremediation and bioaugmentation in their methodology.